In the last year, our work has covered several major areas: I. Tec Kinases: Mutations affecting the Tec kinase, Btk, cause the genetic disorder X-linked Agammmaglobulimemia, characterized by abnormal B cell development and function. Over the last 15 years, we showed that the Tec kinases expressed in T lymphocytes, Itk and Rlk, are important modulators of T cell signaling: mutations of Itk and Rlk do not prevent T cell development and function, but alter outcomes by affecting T cell receptor signaling strength via activation of PLC-g and Ca++ mobilization. Confirming its importance, mutations of Itk have been described in a profound EBV-induced lethal immunodeficiency. Recently, we have focused on the effects of Itk on cytotoxic T lymphocyte (CTL) function, which is critical for clearing viral infection. We have demonstrated that Itk is critical for killing by CTLs, being required both for full activation of CD8 T cells and for the final stages of degranulation of cytotoxic granules required for killing cellular targets (Kapnick et al, J. Immunol 2017). In related work, we used a variety of T cell receptor transgenic and mutant mice to probe requirements for terminating cytotoxic activity against a target, which may allow CTLs to proceed on and kill other targets, a process known as serial killing (Ritter, Kapnick et al, PNAS 2017). Most recently, we have now developed a series of conditional Itk-mutant mice, including ones that can be inhibited acutely by designer inhibitors (Gomez-Rodriguez, in progress), providing potential models for Itk-based therapeutics. II. Phosphoinositide 3 Kinase (PI3K) delta: As part of a collaborative study, we previously helped describe and characterize activating mutations affecting PI3Kdelta, a hematopoietic-specific member of the PI3K catalytic subunit in patients with sino-pulmonary infections, mucosal lymphoid nodules, decreased circulating lymphocytes, lymphoproliferation, and EBV viremia. Our work focused on characterization of CD8+ cell defects in these patients, which showed elevated activation of downstream PI3K targets, including increased pAKT, and mTOR downstream targets (Lucas et al, Nature Immunol. 2014). To further understand these defects, we have generated a mouse model and are using these mice to provide new insight into the requirements for PI3K in immune homeostasis and function. Remarkably, these mice recapitulate multiple features of the disease. We are using them to dissect T and B cell-intrinsic components to these phenotypes, including autoimmunity, and to evaluate therapeutic approaches to this disease (Preite et al and Cannons et al, in progress). III. Regulation of Tfh cells and humoral immune responses: Another major focus of our work is SAP, mutations of which cause the genetic disorder X-linked proliferative syndrome (XLP1),characterized by fatal EBV-infection, lymphomas, and antibody defects. Using gene-targeted mice we generated (Czar et al 2001), we showed that SAP-/- T cells failed to provide essential signals for B cells to generate germinal centers and long-term antibody responses, the hallmarks of successful vaccination. This work has helped define the importance of a subset of T cells, Tfh cells, that are required to provide signals for B cells to form germinal centers (Qi et al, Nature, 2008; Cannons et al, Immunity, 2010). Our work has provided insight into the requirement for T:B cell interactions in the development and function of Tfh cells (Lu et al Immunity 2011), the critical helper T cell population required for providing signals to B cells for germinal center formation and long-term humoral immunity, a key feature of protective responses to most immunizations. More recently, we have used RNAseq to evaluation Tfh-specific gene-expression signatures. We found that the transcription factor TCF1, a component of the Wnt signaling pathway, is selectively expressed in Tfh cells in response to viral infection. Using conditional knockout mice and shRNA knockouts, we and others recently provided evidence that TCF1 is required for Tfh responses to viral infection (Wu et al, Cell Reports, 2015). Our work helps provide insight into the regulation of this important T helper cell population, which permits an organism to respond appropriately to distinct infectious organisms and vaccines (Cannons et al Trends Immunol. 2013). In recent work, we have found that similar signaling and transcriptional networks are required for long-term CD8 cell responses to chronic infection, and that TCF1 marks a population of stem-like CD8 cells with remarkable transcriptional and metabolic overlap with Tfh cells (Wu et al, Sci Immunol 2016).We are now evaluating their function in responses to tumors in mice and humans. IV. To increase our ability to probe the immune system, we have developed new CRISPR mediated tools to inactivate multiple genes in mice and in primary T cells (Huang et al PLOS One 2016 and in progress). We are using these tools to probe function of the SLAM family members and of other genes involved in Tfh cell differentiation and in lymphocyte adhesion (Huang et al, in progress and Johansen et al, In progress). V. To further probe signaling in the immune system in response to infection, we have generated Gcamp6 Ca2+ reporter mice (Mosemann et al, Sci Immunol. 2016).